Human IFN gamma receptor cytoplasmic domain: expression and interaction with HuIFN gamma.
To investigate the structural basis for human interferon gamma (huIFN gamma) binding to intracellular regions of the human IFN gamma receptor (huIFN gamma R), we have subcloned and expressed the huIFN gamma R free of fusion proteins in the yeast strain Pichia pastoris. HuIFN gamma bound to the cytoplasmic domain of the receptor via the IFN gamma C-terminus. Binding was inhibited by both human and mouse C-terminus peptides. N-terminus peptides failed to inhibit cytoplasmic binding. Thus, while extracellular receptor domain binding is species specific, binding to the cytoplasmic domain of the receptor is species non-specific. In solid-phase binding assays, IFN gamma had a Kd of 3.7 x 10(-8) M for the newly expressed cytoplasmic domain. Peptide competitions showed that IFN gamma bound to a receptor site corresponding to the membrane proximal residues 253-287, which is adjacent to the site of binding of the tyrosine kinase JAK2. The cytoplasmic binding affinity and binding site specificity suggest that the huIFN gamma R cytoplasmic domain can function independent of the extracellular domain to bind huIFN gamma and induce the biological activity previously associated with internalized huIFN gamma.
Green
M M
MM
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA. mgreen@micro.ifas.ufl.edu
Larkin
J
J
Subramaniam
P S
PS
Szente
B E
BE
Johnson
H M
HM
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D017471:Receptors, Interferon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D000096743:Interferon gamma Receptor
D017931:DNA Primers; D007371:Interferon-gamma; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D000096743:Interferon gamma Receptor